The capsular antigens of Escherichia coli can be divided into group 1 and group 2 serotypes. It was previously thought that a single strain could possess only a single serotype. The biologic role of group 1 capsules has yet to be established. The majority of isolates of Escherichia coli responsible for extra-intestinal infections possess group 2 capsules. The majority are non-K1, but only the K1 antigen is widely held to be an important virulence factor. We are studying the non-K1 blood isolated (CP9, 04/K54/H5) as a model pathogen and have established that its K54 capsular polysaccharide is an important virulence factor in vitro and in vivo. The genes responsible for synthesizing several group 2 capsular polysaccharides have been cloned and their functional organization established. However, neither genes nor proteins involved in the regulation of group 2 capsule synthesis have yet been identified. In contrast, the regulation of the group 1 capsules produced by Escherichia coli K-12 strains, the K30 serotype and other members of the Enterobacteriaceae genera have been extensively studied. RcsA, RcsB and RcsF have been identified as positive regulators and RcsC and Lon as negative regulators. Therefore, we began an evaluation of the regulation of the group 2, K54 capsular polysaccharide. As starting point, we assessed the role of the group 1 capsule regulators RcsA, RcsB and Lon on the regulation of group 2 capsule production. Our studies have established that CP9 is capable of producing a group 1 capsule. RcsA, RcsB and Lon are present in this K54 background and regulate group 1 capsule expression in a fashion similar to that described for K-12 strains. Two independent group 2 capsule gene protein fusions (TnphoA) were used to evaluate the effects of these regulators on Group 2, K54 capsule production. The results established RcsA as a negative regulator of the group 2, K54 capsular polysaccharide. Its action appears to be mediated through RcsB and is greatest at 28 degrees centigrade, but decreases at higher temperatures. Furthermore, the results also suggest the existence of another Lon sensitive, negative regulator of group 2, K54 capsule production, which is active at higher temperatures.